1. Field of the Invention
The present invention relates to nucleotide sequences encoding a modulator of NF-xcexaB, and to the polypeptides encoded by the nucleotide sequences. In particular, the invention relates to nucleotide sequences and the polypeptides encoded thereby, wherein the polypeptides are involved in the response to NF-xcexaB activating stimuli, including HTLV-I Tax, LPS, PMA and IL-1.
2. Discussion of the Background
The Rel/NF-xcexaB family of transcription factors plays important roles in immune and stress responses, in inflammation, in apoptosis, and regulates the expression of numerous cellular and viral genes (for recent reviews, see Baldwin, 1996; Verma et al., 1995; May and Ghosh, 1998). The NF-xcexaB activity is composed of homo- or heterodimers of related proteins that share a conserved DNA-binding and dimerization domain called the Rel Homology Domain. In most cell types, NF-xcexaB is sequestered in the cytoplasm bound to inhibitory proteins called IxcexaB-xcex1, IxcexaB-xcex2 and IxcexaB-xcex5. In response to diverse stimuli, including inflammatory cytokines, mitogens, bacterial lipopolysaccharide (LPS), or some viral products, active NF-xcexaB is released and translocated to the nucleus as a result of the proteolytic degradation of IxcexaB proteins. Phosphorylation of IxcexaBxcex1 on Ser 32 and 36 targets the molecule for degradation by the ubiquitin-26S proteasome pathway. While the processes leading to the degradation of the IxcexaB proteins are relatively well understood, the mechanism by which a variety of distinct signals initiated from the cell membrane are transduced to their common targets, the IxcexaB proteins, remains to be elucidated. A protein kinase activity was identified as a large multisubunit complex which can phosphorylate IxcexaBxcex1 at Ser 32 and 36 (Chen et al., 1996; Lee et al., 1997). Most recently, two related kinases have been cloned which contain a catalytic domain at the amino-terminus and a leucine zipper (LZ) as well as a helix-loop helix (HLH) motif at the carboxy terminus (Didonato et al., 1997; Mercurio et al., 1997; Regnier et al., 1997; Woronicz et al., 1997; Zandi et al., 1997). Although both of them have been shown to be essential contributors to cytokine-mediated NF-xcexaB activation, understanding of the precise nature of the IxcexaB kinase activity and its regulatory mechanisms needed further investigation and identification of the other subunits of the kinase complex. Another important issue unanswered was how discrete activation signals triggered by a variety of known stimulators are integrated to give rise to IxcexaB kinase activity.
One attractive approach to such questions would be the use of somatic cell genetics. Although the diploidy of the mammalian genome presents a major hurdle to a genetic approach, successful establishment of recessive mutants has provided helpful information on a signaling pathway and a reliable way to identify relevant gene(s) by complementation. Indeed, the Janus kinase family of tyrosine kinases was identified as essential signal transducers for the interferons through a genetic approach (Darnell et al., 1994; Velazquez et al., 1992). Concerning the NF-xcexaB signaling pathways, the characterization of a mutant of the murine pre-B cell line 70Z/3, 1.3E2, has previously been reported, which had been isolated by selecting cells unable to express surface IgM following lipopolysaccharide stimulation (Courtois et al., 1997).
The recent description of a high molecular weight cytoplasmic complex able to phosphorylate IxcexaBxcex1 on serines 32 and 36 (Chen et al., 1996; Lee et al., 1997) has prompted intense studies, which culminated a few months ago with the cloning of two kinases, named IKK-1 and IKK-2, or IKKxcex1 and IKKxcex2 (Didonato et al., 1997; Mercurio et al., 1997; Regnier et al., 1997; Woronicz et al., 1997; Zandi et al., 1997). Two approaches were used to this end: one involved biochemical purification from a cytoplasmic extract derived from TNF-treated HeLa cells (Didonato et al., 1997; Mercurio et al., 1997; Zandi et al., 1997), while the other used a 2-hybrid screen using as a bait NIK, a protein kinase previously shown to be involved in TNF- and IL-1-induced NF-xcexaB activation (Regnier et al., 1997; Woronicz et al., 1997). The cloned kinases were postulated to directly phosphorylate serines 32 and 36 of IxcexaBxcex1, although this has not been formally demonstrated. The reason for this uncertainty is that all kinase assays reported so far rely on immunoprecipitation of transfected or in vitro translated IKK, therefore leaving open the possibility that the xe2x80x9ctruexe2x80x9d IxcexaB kinase is coprecipitated together with IKK and the rest of the high molecular weight complex. Immunoprecipitation of one kinase from extracts of cells transfected with the two kinases results in the coprecipitation of the second kinase, and a more detailed study has demonstrated that hetero-association was favored over homo-association. The sequence of IKK-1 and IKK-2 has revealed two interesting features: a leucine zipper and a helix-loop-helix motif. Deletion of the LZ in one of the kinases results in the abrogation of coimmunoprecipitation with either itself or the other kinase, and a strong reduction in the resulting kinase activity. However it is unclear whether the LZ motif is required for direct interaction between the kinase subunits or between the kinase(s) and some other component of the complex. Deletion of the HLH motif leaves the coimmunoprecipitation of the two kinases intact, but strongly reduces the resulting kinase activity. In the assays used in the above mentioned papers, transfected IKK-2 seems to exhibit a stronger basal kinase activity when compared to IKK-1 (Mercurio et al., 1997; Zandi et al., 1997). Zandi et al. (Zandi et al., 1997) also observed that cotranslation of the two kinases in wheat germ extracts resulted in no IxcexaB kinase activity, suggesting that either post-translational modifications or additional components of the complex (or both) are required. Cotranslation of the two kinases in wheat germ extracts precluded their association. One possibility is that the kinase subunits need to be incorporated into the 600-800 kD complex in order to be fully active, and that some critical components of the complex are absent in wheat germ extracts. In any case all these data emphasize the importance of identifying additional components of the complex.
If the identity of molecules involved in NF-xcexaB activation were known, one could block NF-xcexaB activation, and thereby treat cellular dysfunctions associated therewith, by inactivating these molecules.
In view of the aforementioned limited information regarding molecules involved in NF-xcexaB activation, it is clear that there exists a need in the art for identifying the sequences encoding such molecules.
Accordingly, one object of this invention is to provide a modulator of NF-xcexaB and its subunits in purified form that exhibits certain characteristics and activities associated with inhibition of NF-xcexaB activity.
It is a further object of the present invention to provide antibodies to the modulator of NF-xcexaB and its subunits, and methods for their preparation, including recombinant means.
It is a further object of the present invention to provide a method for detecting the presence of the modulator of NF-xcexaB and its subunits in mammals in which invasive, spontaneous, or idiopathic pathological states are suspected to be present.
It is a further object of the present invention to provide a method and associated assay system for screening substances such as drugs, agents and the like, potentially effective in either mimicking the activity or fighting against the adverse effects of the modulator of NF-xcexaB and/or its subunits in mammals.
It is a still further object of the present invention to provide a method for the treatment of mammals to control the transcriptional activity induced by NF-xcexaB, so as to alter the adverse consequences of such presence or activity, or where beneficial, to enhance such activity.
It is a still further object of the present invention to provide a method for the treatment of mammals to control the amount or activity of the transcriptional activity of NF-xcexaB, so as to treat or avert the adverse consequences of invasive, spontaneous or idiopathic pathological states.
It is a still further object of the present invention to provide pharmaceutical compositions for use in therapeutic methods which comprise or are based upon the modulator of NF-xcexaB, its subunits, their binding partner(s), or upon agents or drugs that control the production, or that mimic or antagonize the activities of the modulator of NF-xcexaB.
With the foregoing and other objects, advantages and features of the invention that will become hereinafter apparent, the nature of the invention may be more clearly understood by reference to the following detailed description of the preferred embodiments of the invention and to the appended claims.